Creatinine is the naturally occurring breakdown product of creatine. Creatine is an organic acid that is synthesized in the liver and kidneys from arginine, glycine, and methionine (Brosnan J T et al., Ann Rev Nutr 27: 241-261 (2007); Greenhaff P, J Nutr Biochem 8: 610-618 (1997); Wallimann et al., Amino Acids 40: 1271-1296 (2011)). In the kidneys arginine and glycine undergo an aminotransferase producing guanidinoacetic acid that acquires a methyl group from methionine during methytransferase in the liver (Brosnan J T et al., Ann Rev Nutr 27: 241-261 (2007); Greenhaff P, J Nutr Biochem 8: 610-618 (1997); Wyss M et al., Physiol Rev 80: 1107-1213 (2000)). The majority (e.g., 95%) of all creatine in the body is found in skeletal muscle, and plays a pivotal role in energy homeostasis (Greenhaff P, J Nutr Biochem 8: 610-618 (1997); Williams M H et al., J Am Coll Nutr 17: 216-234 (1998)). In the muscle, creatine converts into phosphocreatine by creatine kinase, which is utilized to increase the ATP pool during explosive movements such as wrestling or jumping. Creatine is non-enzymatically converted into creatinine (Wyss M et al., Physiol Rev 80: 1107-1213 (2000); Santos R V, et al. Life Sci 75: 1917-1924 (2004)). Creatinine diffuses out of the muscle into the blood and is excreted in the urine. Blood creatinine levels are used as an indicator of renal function. For instance, 50-100 μM of creatinine in the blood is considered normal (Brosnan J T et al., Ann Rev Nutr 27: 241-261 (2007); McDonald T L et al., J Antibiot (Tokyo) 65: 1:53-156 (2012)). Creatinine has thought to have been an inert waste product with no active function; however, recent studies have challenged this dogma.
Previous works studying the potential of creatinine as an immunomodulator have found that creatinine (and its derivatives such as creatinine HCl) possesses biological activity. Indeed, one study conducted by Madan et al. (1979) involved injecting rats with different doses of creatinine and a variety of inflammatory agents (e.g., 5-hydroxytryptamine creatine sulfate, nystatin, and carrageen) to induce acute or chronic inflammation (Indian J Physiol Pharmacol 1-7). Edema was measured in all rats and was decreased with the presence of creatinine. In this study, creatinine acted as an anti-inflammatory agent against acute and chronic inflammation in rats. Another study done by Leland et al. (2011), examined the effects of creatine and creatinine HCl on molecules associated with recognizing pathogen-associated molecular patterns (“PAMPs”) in mouse macrophages (Int Immunopharm 11: 1341-1347). In this study, mRNA levels were assayed for four toll-like receptors (TLRs)—TLR-2, TLR-3, TLR-4, and TLR-7. Transcript levels for all four TLRs were reduced following exposure of the cells to creatinine HCl. No alterations in cell death were observed. This study suggested that creatinine may have the ability to dampen the innate immune response.
Furthermore, another study demonstrated the ability of creatinine HCl to suppress bacterial replication. Addition of creatinine HCl to a growth medium decreased a wide array of Gram negative and Gram positive bacteria as well as drug resistant Staphylococcus aureus (“MRSA”) and vancomycin resistant Enterococcus faecium (“VRE”). Although the mechanism was not clearly defined, the addition of a proton pump inhibitor decreased the concentration of creatinine HCl necessary to kill cells, suggesting the mechanism of action was related to the bacterial cells' capacity to pump out protons (McDonald T L et al., J Antibiot (Tokyo) 65: 153-156 (2012)). A subsequent study from the same group showed that creatinine HCl does not affect the growth of fungi; an observation that was used to demonstrate that creatinine HCl could be used as a novel additive in fungal growth media to permit fungi to grow efficiently without bacterial contamination (Smithee et al., J Microbiol Meth 105: 155-161 (2014)). Such an effect could aid in the identification of novel antibiotic-producing fungi from environmental sources.
Collectively, these studies indicate creatinine is not an inert waste product of creatine, and instead has an active function particularly when protonated. However, these studies present only general findings. The full effects of creatinine remain unknown and are not commonly understood by those of ordinary skill in the art. Accordingly, there remains a need in the art to further understand and appreciate how creatinine works within the body and the effects that may result therefrom.